Summary Osteoarthritis (OA) affects an estimated 27 million people in the United States and is characterized by joint pain and cartilage loss. One of the risk factors for developing OA is having particular genetic variations at certain locations in the genome, such as a specific single nucleotide polymorphism in the regulatory region of the growth and differentiation factor 5 (GDF5) gene. The mechanism by which a causal variant increases the risk for OA is unclear, partly because there is substantial genetic variation among OA patients and lifestyle differences also affect the development of OA. The production of cartilage tissue that is perfectly matched except for the causal variant in question would allow for insight into how a specific genetic variant affects the response of cartilage to OA stimuli such as inflammatory cytokines. Additionally, it would provide an important tool for in vitro drug screens that seek to identify candidate drugs optimized for patients with particular genetic profiles. We propose to develop a novel in vitro system for studying the functional effect of identified OA causal variants on the biochemical and mechanical properties of articular cartilage using genome editing of induced pluripotent stem cells (iPSCs) and cartilage tissue engineering. Our hypothesis is that user-defined precise genetic changes in iPSCs before subsequent chondrogenic differentiation will alter the cartilage production and render the tissue more susceptible to the pro-inflammatory cytokine interleukin-1 alpha (IL-1?), which has been implicated in the pathogenesis of OA. Genome editing will be performed with engineered nucleases targeted to specific loci to stimulate gene targeting by homologous recombination. We will analyze the engineered cartilage for both biochemical composition and mechanical properties. Furthermore, we will also use RNA-Seq to detect how the genetic changes affect global gene expression. Our first aim will be to pursue the T/C single nucleotide polymorphism rs143383 of GDF5 as an initial example of this generally applicable technique. Our second aim will be to make cartilage that represents five different variants in a region of the genome that has been associated with an increased risk for OA by a genome-wide association study. One goal of this aim will be to determine which of the genetic changes is responsible for the increased OA risk. Together, this work will establish a rigorous in vitro system for testing the effect of particular genetic variants on OA development. This knowledge has the potential to provide a platform technology for discovering OA therapeutics that are matched to patients with particular genetic risk factors.